FURTHER CHARACTERIZATION OF THE PORCINE ZONA-PELLUCIDA ANTIGEN CORRESPONDING TO MONOCLONAL-ANTIBODY 3A4-2G1 EXCLUSIVELY CROSS-REACTIVE WITH PORCINE AND HUMAN ZONAE PELLUCIDAE

Biochemical and molecular analyses were performed for the porcine zona pellucida (ZP) antigen corresponding to the monoclonal antibody (Mab) 3A4-2G1 exclusively cross-reactive with porcine and human zonae pellucidae (ZPe). By SDS-PAGE and Western blot analysis, Mab 3A4-2G1 reacted with a 92-kDa component of solubilized porcine ZP (s-PZP) under non-reducing conditions, while reacting with a 23-kDa component under reducing conditions. Diagonal SDS-PAGE revealed that the 92-kDa component was composed of 69 kDa and 23 kDa molecules. In two-dimensional O'Farrell's electrophoresis, the antigen molecules reactive to Mab 3A4-2G1 were distributed over a wide range of PI as a family of proteins around 92 kDa and 23 kDa regions under non-reducing and reducing conditions, respectively. A murine antiserum raised to the isolated 23-kDa component reacted not only with the 23-kDa but also with the 92-kDa glycoprotein family. The anti-23 kDa serum showed a strong inhibitory effect on human sperm-egg interaction by in vitro fertilization. The reactivity of s-PZP to Mab 3A4-2G1 was markedly reduced by treatments with proteolytic enzymes but not significantly with glycosidic enzymes. However, chemical deglycosylation of the antigen markedly reduced the antigen's activity. These results suggested that Mab 3A4-2G1 recognized a conformational antigen epitope structure of ZP.